Exploring the links between peptoid antibacterial activity and toxicity.

Peptoids are a promising class of antimicrobial agents with reported activities against a range of both Gram-positive and Gram-negative bacteria, fungi and most recently parasites. However, at present the available toxicity data is somewhat limited and as such rationally designing effective antimicrobial peptoids can be challenging. Herein, we present the toxicity profiling of a series of linear peptoids against mammalian cell lines (HaCaT and HepG2). The cytotoxicity of the peptoid library has then been correlated with their antibacterial properties against Gram-positive and Gram-negative bacteria and also to the hydrophobicity of the peptoid sequences. The work presented provides valuable data to aid in the future rational design of antimicrobial peptoids.

Multifaceted role of hair follicle dermal cells in bioengineered skins.

BACKGROUND: The method of generating bioengineered skin constructs was pioneered several decades ago; nowadays these constructs are used regularly for the treatment of severe burns and nonhealing wounds. Commonly, these constructs are comprised of skin fibroblasts within a collagen scaffold, forming the skin dermis, and stratified keratinocytes overlying this, forming the skin epidermis. In the past decade there has been a surge of interest in bioengineered skins, with researchers seeking alternative cell sources, or scaffolds, from which constructs can be established, and for more biomimetic equivalents with skin appendages. OBJECTIVES: To evaluate whether human hair follicle dermal cells can act as an alternative cell source for engineering the dermal component of engineered skin constructs. METHODS: We established in vitro skin constructs by incorporating into the collagenous dermal compartment: (i) primary interfollicular dermal fibroblasts, (ii) hair follicle dermal papilla cells or (iii) hair follicle dermal sheath cells. In vivo skins were established by mixing dermal cells and keratinocytes in chambers on top of immunologically compromised mice. RESULTS: All fibroblast subtypes were capable of supporting growth of overlying epithelial cells, both in vitro and in vivo. However, we found hair follicle dermal sheath cells to be superior to fibroblasts in their capacity to influence the establishment of a basal lamina. CONCLUSIONS: Human hair follicle dermal cells can be readily interchanged with interfollicular fibroblasts and used as an alternative cell source for establishing the dermal component of engineered skin both in vitro and in vivo.

The role of activins and follistatins in skin and hair follicle development and function.

Investigations of the signalling between epithelial and mesenchymal compartments of skin during hair follicle initiation in utero and hair cycling have revealed the importance of the TGFbeta superfamily in ectodermal organogenesis and morphogenesis. In particular the activins, their receptors and binding proteins such as follistatin, have been shown to be important regulators of cell proliferation, differentiation and apoptosis in hair follicle initiation, hair cycling, normal skin homeostasis and wound healing. Transgenic mice lacking various components of the activin signalling pathways display varying ectodermal pathologies including altered pelage hair follicle initiation. This review summarises the activin signal transduction pathways and the interactions between activins and other TGFbeta signalling systems during hair follicle formation, hair growth cycling, skin function and wound healing.

Plasticity of hair follicle dermal cells in wound healing and induction.

The capacity of adult hair follicle dermal cells to participate in new follicle induction and regeneration, and to elicit responses from diverse epithelial partners, demonstrates a level of developmental promiscuity and influence far exceeding that of interfollicular fibroblasts. We have recently suggested that adult follicle dermal cells have extensive stem or progenitor cell activities, including an important role in skin dermal wound healing. Given that up to now tissue engineered skin equivalents have several deficiencies, including the absence of hair follicles, we investigated the capacity of follicle dermal cells to be incorporated into skin wounds; to form hair follicles in wound environments; and to create a hair follicle-derived skin equivalent. In our study, we implanted rat follicle dermal cells labelled with a vital dye into ear and body skin wounds. We found that they were incorporated into the new dermis in a manner similar to skin fibroblasts, but that lower follicle dermal sheath also assimilated into hair follicles. Using different combinations of follicle dermal cells and outer root sheath epithelial cells in punch biopsy wounds, we showed that new hair follicles were formed only with the inclusion of intact dermal papillae. Finally by combining follicle dermal sheath and outer root sheath cells in organotypic chambers, we created a skin equivalent with characteristic dermal and epidermal architecture and a normal basement membrane - the first skin to be produced entirely from hair follicle cells. These data support the hypothesis that follicle dermal cells may be important in wound healing and demonstrate their potential usefulness in human skin equivalents and skin substitutes. While we have made progress towards producing skin equivalents that contain follicles, we suggest that the failure of cultured dermal papilla cells to induce follicle formation in wounds illustrates the complex role the follicle dermis may play in skin. We believe that it demonstrates a genuine dichotomy of activity for follicle cells within skin.

Emulsion-derived foams (PolyHIPEs) containing poly(epsilon-caprolactone) as matrixes for tissue engineering.

The preparation of PolyHIPE foams containing poly(epsilon-caprolactone) from macromonomers by free radical homo- or copolymerization is described. The macromonomers are synthesized from PCL diols and are polymerized in the continuous phase of high internal phase emulsions (HIPEs). Subsequent drying yields low-density foams with cell diameters of 5-100 microm. Foam morphology, as determined by scanning electron microscopy, depends on the type of diluent (styrene, methyl methacrylate, or toluene) added to the emulsion organic phase and on the PCL content. Increasing the latter increases the continuous phase viscosity to a point where emulsion formation is impeded. Foam swelling in toluene, 2-propanol, and water was investigated by solvent imbibition and increased with increasing solvent hydrophobicity. Furthermore, it was found generally to decrease with increasing PCL content, due to increasing cross-link density. Swelling generally increased when higher molar mass PCL macromonomer was used due to the formation of a less tightly cross-linked network. One type of foam sample was shown to support the growth of human fibroblasts over a period of 2.5 days.

Hair follicle dermal sheath cells: unsung participants in wound healing.

The dermal sheath that surrounds the outside of the hair follicle contains progenitor cells that maintain and regenerate the dermal papilla, a key component for hair growth. Our contention is that dermal sheath cells have other roles. We believe that they can become wound healing fibroblasts and perform an important function in the repair of skin dermis after injury. The dermal sheath has close developmental and anatomical parallels with follicle outer root sheath, the epithelial component that contains the stem cells responsible for replacing skin epidermis. Dermal sheath cells also have a myofibroblast or wound healing phenotype, and in animals with high follicle densities differences in wound healing are observed in conjunction with changes in the hair growth cycle. Similarly, in human beings there are apparent differences in wound healing responses between hairy and non-hairy body sites. Moreover, clinical and experimental data suggest that the involvement of follicle-derived dermal cells results in qualitatively improved dermal repair. Therefore, in a therapeutic context, hair follicle dermal cells provide an accessible option for the creation of dermal or full skin equivalents that could both improve wound healing and reduce scarring. Indeed, given the inductive properties of adult hair follicle dermal cells, it is reasonable to envisage a tissue engineering approach for the production of a skin equivalent that will grow hair follicles when grafted.

Hair follicle predetermination.

Recent genetic and molecular studies of hair follicle (HF) biology have provided substantial insight; however, the molecular data, including expression patterns, cannot be properly appreciated without an understanding of the basic cellular rearrangements and interactions that underpin HF cyclic transformations. We present a novel interpretation of the major cellular processes that take place during HF cycling--the hypothesis of hair follicle predetermination. This hypothesis is an extension of previous models of HF cellular kinetics but has two critical modifications: the dual origin of the cycling portion of the HF, and the timing of the recruitment of stem cells. A compilation of evidence suggests that the ascending portion of the HF (hair shaft and inner root sheath) arises not from bulge-located HF stem cells that contribute to the formation of only the outer root sheath (ORS), but instead from the germinative cells localized in the secondary hair germ. In middle anagen, upon completion of the downward growth of the HF, cells derived from the bulge region migrate downward along the ORS to reside at the periphery of the HF bulb as a distinct, inactive cell population that has specific patterns of gene expression - 'the lateral disc'. These cells survive catagen-associated apoptosis and, under the direct influence of the follicular papilla (FP), transform into the hair germ and acquire the ability to respond to FP signaling and produce a new hair. Thus, we propose that the specific sensitivity of germ cells to FP signaling and their commitment to produce the ascending HF layers are predetermined by the previous hair cycle during the process of transformation of bulge-derived lateral disc cells into the secondary hair germ.

In vivo induction of hair growth by dermal cells isolated from hair follicles after extended organ culture.

Successful hair follicle organ culture has been established for some time, but hair growth in vitro is limited and generally terminates prematurely in comparison with in vivo. The reasons why growth stops in culture are as yet unknown. In this investigation, adult rat vibrissa follicles for which growth in culture is limited to about 10 d, were maintained in vitro for a minimum of 20 d after the hair shaft stopped growing. The pattern of fiber growth and long-term follicle pathology reflected the initial hair cycle stage at the time of isolation. Furthermore, there was evidence that a group of follicles put into culture when in late anagen were attempting to cycle in vitro. Microscopy showed that, in spite of widespread pathologic changes to the follicle epithelium, dermal cells in the follicle showed remarkable resilience. Their viability was confirmed when primary cell cultures were established from isolated dermal tissue. These cells labeled positively for alpha-smooth muscle actin, an established marker of hair follicle dermal cell phenotype in vitro. Moreover, isolated dermal tissue induced hair growth when implanted into inactivated hair follicles in vivo. These data confirm that the cessation in hair growth is not due to a loss of the inductive capacity in the dermal component. Long-term organ culture may provide opportunities to investigate factors that are expressed or lost during hair growth cessation. In addition it may be possible to develop this method further to obtain a reliable and predictable model of hair follicle cycling in vitro.

Trans-species hair growth induction by human hair follicle dermal papillae.

A series of experimental bioassays has shown that the dermal papilla of the adult rodent vibrissa hair follicle retains unique inductive properties. In view of the many phenotypic and functional differences between specific hair follicle types, and the growing interest in hair follicle biology and disease, it remains important to establish that the human hair follicle dermal papilla has equivalent capabilities. In this study we tested the ability of human hair follicle papillae to induce hair growth when implanted into transected, athymic mouse vibrissa follicles. The implanted papillae that interacted with mouse follicle epithelium created new fibre-producing follicle end bulbs. The origin of the papillae in the recombinant structures was confirmed using laser capture microdissection and human specific gender determination by PCR. The demonstration that intact adult human dermal papillae can induce hair growth has implications for molecular analysis of basic hair growth mechanisms, particularly since the study involved common epithelial-mesenchymal signalling and recognition properties across species. It also improves the prospects for a cell-based clinical approach to hair follicle disorders.

A correlation between versican and neurofilament expression patterns during the development and adult cycling of rat vibrissa follicles.

Versican, a proteoglycan recently implicated in hair follicle induction, has been shown to influence axon outgrowth in vitro and in vivo. We used immunohistochemistry to study the relationship between versican expression and innervation, during rat vibrissa follicle development and the adult hair cycle. During development, nerve fibres were commonly associated with areas of weak versican expression, and the path of axons appeared to be delineated by sharp boundaries of versican expression. Versican expression changed in the lower follicle dermis during the adult hair follicle cycle but remained strong around the follicle neck reflecting the constant innervation. Our observations show a correlation between versican expression and peripheral innervation indicating that versican may have a dual role in hair follicle biology.

Adult corneal epithelium basal cells possess the capacity to activate epidermal, pilosebaceous and sweat gland genetic programs in response to embryonic dermal stimuli.

Recent work has shown remarkable plasticity between neural and hematopoeitic, as well as between hematopoeitic and muscle stem cells, depending on environmental stimuli (Fuchs, E. and Segre, J. A. (2000) Cell 100, 143-155). Stem cells give rise to a proliferative transient amplifying population (TA), which is generally considered to be irreversibly committed. Corneal epithelium provides a particularly useful system for studying the ability of TA cells to activate different genetic programs in response to a change in their fibroblast environment. Indeed, corneal stem and TA cells occupy different localities - stem cells at the periphery, and TA cells more central (Lehrer, M. S., Sun, T. T. and Lavker, R. M. (1998) J. Cell Sci. 111, 2867-2875) - and thus can be discretely dissected from each other. It is well known that pluristratified epithelia of cornea and skin display distinct programs of differentiation: corneal keratinocytes express keratin pair K3/K12 and epidermal keratinocytes keratin pair K1-2/K10; moreover, the epidermis forms cutaneous appendages, which express their own set of keratins. In our experiments, central adult rabbit corneal epithelium was thus associated either with a mouse embryonic dorsal, upper-lip or plantar dermis before grafting onto nude mice. Complementary experiments were performed using adult mouse corneal epithelium from the Rosa 26 strain. The origin of the differentiated structures were identified in the first case by Hoechst staining and in the second by the detection of beta-galactosidase activity. The results show that adult central corneal cells are able to respond to specific information originating from embryonic dermis. They give rise first to a new basal stratum, which does not express anymore corneal-type keratins, then to pilosebaceous units, or sweat glands, depending of the dermis, and finally to upper layers expressing epidermal-type keratins. Our results provide the first evidence that a distinct TA cell population can be reprogrammed.

Localisation of members of the notch system and the differentiation of vibrissa hair follicles: receptors, ligands, and fringe modulators.

Hair vibrissa follicle morphogenesis involves several cell segregation phases, in the dermis as well as in the epidermis. The expression of Notch-related genes, which are well established mediators of multiple cell segregation events in Drosophila development, was studied by in situ hybridisation during embryonic mouse vibrissa follicle morphogenesis and the first adult hair cycle. The results show that two receptors, Notch1 and -2, three ligands, Delta1, Serrate1, and -2, and the three Fringe regulators, Lunatic, Manic, and Radical, are expressed in different locations and morphogenetic stages. First, the appearance of hair vibrissa primordia involves the expression of complementary patterns of Notch2, Delta1, and Lunatic Fringe in the dermis and of Notch1, Serrate2, and Lunatic Fringe in the epidermis. Second, this expression pattern is no longer found after stage 3 in the dermis. Meanwhile, in the epidermis, the expression of Notch1, Serrate2, and Lunatic Fringe before the formation of the placode may be involved in determining two populations of epidermal cells in the developing follicle. Third, complementary expression patterns for Notch1, Manic, and Lunatic Fringe, as well as Serrate1 and -2 as previously shown (Powell et al., 1998), are progressively established from stage 4 of embryonic development both in the outer root sheath and in the hair matrix. These patterns are consistent with the one found in the adult anagen phase. During the hair vibrissa cycle, Notch1 and Manic Fringe display temporal and spatial changes of expression, suggesting that they may intervene as modulators of trichocyte activities.

Cellular and developmental aspects of androgenetic alopecia.

The hair follicle is a highly complex system that can be investigated at many levels and from multiple perspectives. However, underlying the cyclic production of all hair fibres are a set of common developmental processes. Many current investigations of androgenetic alopecia concentrate on the direct influences of hormones on hair follicles at the cellular or intracellular level. This paper attempts to step back from this and consider the process of terminal to vellus transition in androgenetic alopecia in terms of basic cellular and developmental mechanisms. Ideas about the mechanism and timing of follicle size reduction are put forward, but the paper also tries to point out inherent difficulties in the investigation of androgenetic alopecia and important gaps in current knowledge.

Hair cycle stage of the mouse vibrissa follicle determines subsequent fiber growth and follicle behavior in vitro.

The establishment of culture models representative of all aspects of in vivo hair follicle behavior is an important goal for theoretic and analytic studies. Rodent vibrissa follicles have regular, predictable, and relatively short growth cycles. In this investigation, we took advantage of these properties; we classified mouse vibrissa follicles according to different phases in the hair cycle and then compared fiber growth and morphologic changes in culture. Follicles isolated in the early phase of the growth cycle produced fine growing fibers with an average growth that exceeded 3 mm over 15 d. Even when hair growth had slowed or halted subsequently, histology showed that these follicles retained an anagen-like morphology. By contrast, follicles isolated toward the end of the growing cycle produced thicker fibers for much shorter periods, after which growth ceased and the fibers lifted up from the base of the follicle. Internally, these specimens resembled their telogen counterparts in situ. Follicles isolated in mid-growth demonstrated intermediate fiber growth characteristics. In organ culture, mouse vibrissa follicles therefore closely reflect their in vivo origin in growth characteristics and cycle timing. These data provide new opportunities for studying hair growth cycle mechanisms in vitro, but present a caveat for quantitative studies because there may be a greater growth cycle-related variation than has previously been assumed.

Human hair follicle regeneration following amputation and grafting into the nude mouse.

In this study we investigated the capacity of the human hair follicle to regenerate a fiber-forming bulb after its amputation. We removed the bases from terminal follicles from a variety of sites and transplanted the follicles onto athymic mice, either still attached to a skin graft or as subcutaneous implants of individual follicles. External hair growth was observed on the skin grafts, and histology of the follicles revealed restoration of dermal papillae and follicle bulb structures. This result suggests that the capacity of hair follicles to regenerate their lower structures after removal, which was first demonstrated on whisker follicles, may be a general phenomenon. It emphasizes the importance of specific cellular subpopulations within the follicle and the role of dermal-epidermal interactions in adult follicle activities.

Hair matrix germinative epidermal cells confer follicle-inducing capabilities on dermal sheath and high passage papilla cells.

Low passage cultured dermal papilla cells from adult rats stimulate complete hair follicle neogenesis when re-implanted into heterotypic skin. In contrast, cultured sheath cells are non-inductive despite sharing other behavioural characteristics (a common lineage and in situ proximity) with papilla cells. However, since sheath cells can behave inductively in amputated follicles after regenerating the papilla, this poses the question of what influences the sheath to papilla cell transition? During reciprocal tissue interactions specific epidermal cues are crucial to skin appendage development, and while in vivo assays to date have focussed on dermal interactive influence, our aim was to investigate epidermal potential. We have previously observed that hair follicle epidermal cells display exceptional interactive behaviour when combined with follicle dermal cells in vitro. Thus in the present study, hair follicle germinative, outer root sheath or skin basal epidermal cells were separately combined with each of three non-inductive dermal cell types (high passage papilla, low passage sheath or fibroblast) and then implanted into small ear skin wounds. The sheath/germinative and papilla/germinative cell implants repeatedly induced giant vibrissa-type follicles and fibres. In complete contrast, any single cell type and all other forms of recombination were consistently non-inductive. Hence, the adult germinative epidermal cells enable non-inductive adult dermal cells to stimulate hair follicle neogenesis, effectively, by altering their 'status', causing the sheath cells to 'specialise' and the 'aged' papilla cells to 'rejuvenate'.

Dermal-epidermal interactions. Adult follicle-derived cell populations and hair growth.

Intrinsic dermal-epidermal interactions are central to the development and growth of hair. This article describes investigations into the inductive properties of specific dermal and epidermal cell populations from adult follicles by means of cell culture and in vivo implantation. It highlights the inductive powers of cultured dermal papilla cells and the more recent finding that the germinative epidermal cells of the lower follicle also can stimulate hair growth. How the reconstruction of a hair follicle from its constituent parts has been achieved is described. The significance of these findings is considered with reference to human hair growth, tissue engineering, and the prospects for elucidating the molecular signalling mechanisms that underpin dermal-epidermal interplay.